Novel insect inhibitory proteins

ABSTRACT

Insecticidal proteins exhibiting toxic activity against Coleopteran and Lepidopteran pest species are disclosed, and include, but are not limited to, TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346, TIC4826, TIC4861, TIC4862, TIC4863, and TIC-3668-type proteins. DNA molecules and constructs are provided which contain a polynucleotide sequence encoding one or more of the disclosed TIC3668-type proteins. Transgenic plants, plant cells, seed, and plant parts resistant to Lepidopteran and Coleopteran infestation are provided which contain polynucleotide sequences encoding the insecticidal proteins of the present invention. Methods for detecting the presence of the polynucleotides or the proteins of the present invention in a biological sample, and methods of controlling Coleopteran and Lepidopteran species pests using any of the TIC3668-type insecticidal proteins are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/945,140, filed Nov. 18, 2015 (pending), which application claims thebenefit of priority to U.S. Provisional Application 62/082,504, filedNov. 20, 2014, which are incorporated herein by reference in theirentireties.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing is filed herewith byelectronic submission. The Sequence Listing is incorporated by referencein its entirety, is contained in the file created on Nov. 13, 2015,having the file name “MONS387US_ST25.txt” and which is 117,678 bytes insize (as measured in MS-Windows operating system).

FIELD OF THE INVENTION

The invention generally relates to the field of insect inhibitoryproteins. A novel class of proteins exhibiting insect inhibitoryactivity against agriculturally-relevant pests of crop plants and seedsis disclosed. In particular, the disclosed class of proteins isinsecticidally active against agriculturally-relevant pests of cropplants and seeds, particularly Lepidopteran and Coleopteran species ofinsect pests. Plants, plant parts, and seeds containing a recombinantpolynucleotide construct encoding one or more of the disclosed toxinproteins are provided.

BACKGROUND OF THE INVENTION

Improving crop yield from agriculturally significant plants including,among others, corn, soybean, sugarcane, rice, wheat, vegetables, andcotton, has become increasingly important. In addition to the growingneed for agricultural products to feed, clothe and provide energy for agrowing human population, climate-related effects and pressure from thegrowing population to use land other than for agricultural practices arepredicted to reduce the amount of arable land available for farming.These factors have led to grim forecasts of food security, particularlyin the absence of major improvements in plant biotechnology andagronomic practices. In light of these pressures, environmentallysustainable improvements in technology, agricultural techniques, andpest management are vital tools to expand crop production on the limitedamount of arable land available for farming.

Insects, particularly insects within the order Lepidoptera andColeoptera, are considered a major cause of damage to field crops,thereby decreasing crop yields over infested areas. Lepidopteran pestspecies which negatively impact agriculture include, but are not limitedto, Helicoverpa zea, Ostrinia nubilalis, Diatraea saccharalis, Diatraeagrandiosella, Anticarsia gemmatalis, Spodoptera frugiperda, Spodopteraexigua, Agrotis ipsilon, Trichoplusia ni, Chrysodeixis includens,Heliothis virescens, Plutella xylostella, Pectinophora gossypiella,Helicoverpa armigera, Elasmopalpus lignosellus, Striacosta albicosta andPhyllocnistis citrella. Coleopteran pest species which negatively impactagriculture include, but are not limited to, Agriotes spp., Anthonomusspp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp.,Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnusspp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp.,Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp.,Psylliodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp.,Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.,particularly when the pest is Diabrotica virgifera virgifera (WesternCorn Rootworm, WCR), Diabrotica barberi (Northern Corn Rootworm, NCR),Diabrotica virgifera zeae (Mexican Corn Rootworm, MCR), Diabroticabalteata (Brazilian Corn Rootworm (BZR), Diabrotica undecimpunctatahowardii (Southern Corn Rootworm, SCR) and a Brazilian Corn Rootwormcomplex (BCR) consisting of Diabrotica viridula and Diabroticaspeciosa).

Historically, the intensive application of synthetic chemicalinsecticides was relied upon as the pest control agent in agriculture.Concerns for the environment and human health, in addition to emergingresistance issues, stimulated the research and development of biologicalpesticides. This research effort led to the progressive discovery anduse of various entomopathogenic microbial species, including bacteria.

The biological control paradigm shifted when the potential ofentomopathogenic bacteria, especially bacteria belonging to the genusBacillus, was discovered and developed as a biological pest controlagent. Strains of the bacterium Bacillus thuringiensis (Bt) have beenused as a source for proteins which exhibit pesticidal activity since itwas discovered that Bt strains show a high toxicity against specificinsects. The main feature of Bt's is the production of parasporal bodieswhich contain one or more crystals that contain specific insecticidalendotoxins (Cry proteins) which act upon ingestion by a susceptibleinsect through a pore-forming mechanism of action detrimental for theinsect gut epithelium. Besides Bt, other Bacillus species, such asBacillus sphaericus, and other bacteria species that contain genes thatcontribute to an entomopathogenic phenotype, such as Brevibacilluslaterosporus, have shown potential for pest management.

Insecticidal toxin proteins have been employed in various agriculturalapplications to preserve agriculturally important plants and increaseyields. Insecticidal toxin proteins are used to controlagriculturally-relevant pests of crop plants by mechanical methods, suchas spraying to disperse microbial formulations containing variousbacteria strains onto plant surfaces, and by using genetictransformation techniques to produce transgenic plants and seedsexpressing insecticidal toxin protein.

The use of transgenic plants expressing insecticidal toxin proteins hasbeen globally adapted. For example, in 2012, 26.1 million hectares wereplanted with transgenic crops expressing Bt toxins (James, C., GlobalStatus of Commercialized Biotech/GM Crops: 2012. ISAAA Brief No. 44).The expanded use of transgenic insect-protected crops and the limitednumber of commercially available insecticidal toxin proteins is creatinga selection pressure for alleles that impart resistance to thecurrently-utilized insecticidal proteins. The development of resistancein target pests to insecticidal toxin proteins undermines theeffectiveness and advantages of this technology. Such advantages includeincreased crop yields, reduction in chemical pesticide use, andreduction in the costs and labor associated with chemical pesticide use.

The discovery and development of new forms of insecticidal toxinproteins is central to managing the increase in insect resistance totransgenic crops expressing insecticidal toxin proteins. New proteintoxins with improved efficacy and which exhibit control over a broaderspectrum of susceptible insect species will reduce the number ofsurviving insects which can develop resistance alleles. In addition, twoor more transgenic toxins toxic to the same insect pest and displayingdifferent modes of action in one plant further reduces the probabilityof resistance in a target insect species.

Consequently, there is a critical need to discover and develop effectiveinsecticidal proteins with improved insecticidal properties such asincreased efficacy against a broader spectrum of target insect pestspecies and different modes of action compared to proteins known in theart. A novel protein toxin family from Brevibacillus laterosporus (B.laterosporus) is disclosed in this application along with similar toxinproteins, variant proteins, and exemplary recombinant proteins thatexhibit insecticidal activity against significant target Lepidopteranand Coleopteran pest species, particularly against Western CornRootworm.

SUMMARY OF THE INVENTION

Disclosed herein is a novel group of insect inhibitory recombinantpolynucleotide molecules and polypeptides (toxin proteins) encodedthereby, referred to herein as TIC3668-type proteins, which are shown toexhibit inhibitory activity against one or more pests of crop plants.Each of the proteins can be used alone or in combination with each otherand with other insecticidal proteins and toxic agents in formulationsand in planta, thus providing alternatives to insecticidal proteins andinsecticide chemistries currently in use in agricultural systems.

In one aspect, the invention provides a recombinant polynucleotidemolecule encoding an insect inhibitory polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:25, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, and SEQ ID NO:31. In one embodiment, therecombinant polynucleotide molecule encodes an insect inhibitorypolypeptide comprising at least 35% identity, for instance, at least40%, 50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ IDNO:31. In another embodiment, the recombinant polynucleotide moleculecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64,SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72. In still anotherembodiment the recombinant polynucleotide molecule comprises at least35% identity, for instance, at least 40%, 50%, 60%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleotidesequence selected from the group consisting of SEQ ID NO:37, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, and SEQ ID NO:72. In a further embodiment, the recombinantpolynucleotide molecule comprise a sequence that hybridizes to: (i) thereverse complement of the nucleotide sequence from position 4-885 of asequence selected from the group consisting of SEQ ID NO:37, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, and SEQ ID NO:72; or (ii) the reverse complement a sequenceselected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, and SEQ ID NO:61. In another embodiment, thehybridization conditions are stringent conditions, for instance, suchstringent conditions may comprise hybridization from 4 to 12 hours in50% formamide, 1 M NaCl, and 1% SDS at 37C, and a wash in 0.1×SSC at60C-65C. In further embodiment, the recombinant polynucleotide moleculeis operably linked to a heterologous promoter.

In another aspect, the invention provides an insect inhibitoryrecombinant polypeptide encoded by the recombinant polynucleotidemolecule provided herein. In one embodiment, the insect inhibitoryrecombinant polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQID NO:31. In another embodiment, the insect inhibitory recombinantpolypeptide comprises at least 35% identity, for instance, at least 40%,50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

In a further embodiment, the insect inhibitory recombinant polypeptideexhibits inhibitory activity against an insect species of the orderColeoptera, for instance including Western Corn Rootworm, Southern CornRootworm, Northern Corn Rootworm, Mexican Corn Rootworm, Brazilian CornRootworm, or Brazilian Corn Rootworm complex consisting of Diabroticaviridula and Diabrotica speciosa. In yet a further embodiment, theinsect inhibitory recombinant polypeptide exhibits inhibitory activityagainst an insect species of the order Lepidoptera, for instanceincluding European Corn Borer, Southwestern Corn Borer, Black Cutworm,Fall Army Worm, Corn Earworm, and Soybean Looper.

In yet another aspect, the invention provides a host cell comprising arecombinant polynucleotide molecule of the invention, wherein the hostcell is selected from the group consisting of a bacterial host cell anda plant host cell. In certain embodiments, bacterial host cells includeAgrobacterium, Rhizobium, Bacillus thuringiensis, Brevibacilluslacterosporus, Bacillus cereus, E. coli, Pseudomonas, Klebsiella, andErwinia. In other embodiments, plant cells include an alfalfa, banana,barley, bean, broccoli, cabbage, brassica, carrot, cassava, castor,cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee,corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant,eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine,millets, melons, nut, oat, olive, onion, ornamental, palm, pasturegrass, pea, peanut, pepper, persimmon, pigeon pea, pine, pomegranate,poplar, potato, pumpkin, Radiata pine, radish, rapeseed, rice,rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetcorn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,triticale, turf grass, watermelon, and wheat plant cell.

In a further aspect, the invention provides an insect inhibitorycomposition which may comprise a recombinant polynucleotide molecule ofthe present invention. In one embodiment, the insect inhibitorycomposition may further comprise a nucleotide sequence encoding at leastone other pesticidal agent. In certain embodiments, the at least oneother pesticidal agent is different from the TIC3668-type insectinhibitory polypeptide of the invention and may be selected from thegroup consisting of an insect inhibitory protein, an insect inhibitorydsRNA molecule, and an ancillary protein. In other embodiments, theother pesticidal agent exhibits activity against one or more pestspecies of the orders Lepidoptera, Coleoptera, or Hemiptera. In certainembodiments, the other pesticidal agent is selected from the groupconsisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1B, Cry1C, Cry1D,Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab,Cry3A, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35,Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400,TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901,TIC1201, TIC1415, VIP3A, and VIP3B protein. In yet a further aspect, thepresent invention provides an insect inhibitory composition comprisingan insect inhibitory recombinant polypeptide of the present invention,such as a TIC3668-type insect inhibitory polypeptide, in an insectinhibitory effective amount.

In still another aspect, the invention provides a method of controllinga Coleopteran or Lepidopteran species pest, and controlling aColeopteran or Lepidopteran species pest infestation of a plant, forinstance a crop plant, wherein the method comprises contacting the pestwith an insect inhibitory amount of the insect inhibitory recombinantpolypeptide of the invention, such as a TIC3668-type insect inhibitorypolypeptide.

In a still further aspect, the invention provides a seed comprising arecombinant polynucleotide molecule or insect inhibitory recombinantpolypeptide, such as a TIC3668-type insect inhibitory polypeptide, ofthe invention.

In another aspect, the invention provides a commodity product comprisinga detectable amount of the recombinant polynucleotide molecule, or theinsect inhibitory polypeptide, such as a TIC3668-type insect inhibitorypolypeptide, of the invention. In a further aspect, a commodity productof the invention may comprise a host cell comprising a recombinantpolynucleotide molecule of the invention, wherein the commodity productcomprises a detectable amount of the recombinant polynucleotide moleculeor an insect inhibitory recombinant polypeptide encoded by therecombinant polynucleotide. In certain embodiments, the commodityproducts may include commodity corn bagged by a grain handler, cornflakes, corn cakes, corn flour, corn meal, corn syrup, corn oil, cornsilage, corn starch, corn cereal, and the like, and correspondingsoybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, andvegetable commodity products including where applicable, juices,concentrates, jams, jellies, marmalades, and other edible forms of suchcommodity products containing a detectable amount of suchpolynucleotides and or polypeptides of this application.

In a yet another aspect, the invention provides a method of producingseed comprising the recombinant polynucleotide of the invention, whereinthe method comprises: (a) planting at least one seed comprising therecombinant polynucleotide molecule; (b) growing plants from the seed;and (c) harvesting seed from the plants, wherein the harvested seedcomprises the recombinant polynucleotide molecule.

In a further aspect, the invention provides a recombinant vectorcomprising the recombinant polynucleotide molecule of the invention. Inone embodiment, the recombinant vector is selected from the groupconsisting of a plasmid, a bacmid, a phagemid, and a cosmid.

In another aspect, the invention provides a plant resistant to insectinfestation, wherein the cells of said plant comprise the recombinantpolynucleotide molecule or the insect inhibitory recombinant polypeptideof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the alignment of the collage protein TIC4260 to fiveexemplary TIC3668-type proteins. Positions of sequence diversity arehighlighted in gray shading in this sequence alignment.

FIG. 2 illustrates in planta Western Corn Rootworm (WCR) inhibitoryactivity of exemplary chloroplast targeted and non-targeted maturelength TIC3668-type proteins.

FIG. 3 illustrates in planta WCR inhibitory activity of an exemplarychloroplast targeted and non-targeted mature length TIC-3668-typeprotein.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC3668 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:2 is the amino acid sequence translation of the TIC3668precursor protein from the open reading frame as set forth in SEQ IDNO:1.

SEQ ID NO:3 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC3669 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:4 is the amino acid sequence translation of the TIC3669protein from the open reading frame as set forth in SEQ ID NO:3.

SEQ ID NO:5 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC3670 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:6 is the amino acid sequence translation of the TIC3670precursor protein from the open reading frame as set forth in SEQ IDNO:5.

SEQ ID NO:7 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4076 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:8 is the amino acid sequence translation of the TIC4076precursor protein from the open reading frame as set forth in SEQ IDNO:7.

SEQ ID NO:9 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4078 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:10 is the amino acid sequence translation of the TIC4078precursor protein from the open reading frame as set forth in SEQ IDNO:9.

SEQ ID NO:11 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a collage TIC4260 proteinfrom an open reading frame at nucleotide position 1-951 and atranslation termination codon, created by combining DNA segments fromeach of coding sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7 and SEQ ID NO:9 in-frame to include the sequencevariations from these five different open reading frames.

SEQ ID NO:12 is the amino acid sequence translation of the collageprotein TIC4260 precursor protein from the open reading frame as setforth in SEQ ID NO:11.

SEQ ID NO:13 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4346 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:14 is the amino acid sequence translation of the open readingframe as set forth in SEQ ID NO:13.

SEQ ID NO:15 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4826 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:16 is the amino acid sequence translation of the open readingframe as set forth in SEQ ID NO:15.

SEQ ID NO:17 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4861 protein from anopen reading frame at nucleotide position 1-918 and a translationtermination codon.

SEQ ID NO:18 is the amino acid sequence translation of the open readingframe as set forth in SEQ ID NO:17.

SEQ ID NO:19 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4862 protein from anopen reading frame at nucleotide position 1-945 and a translationtermination codon.

SEQ ID NO:20 is the amino acid sequence translation of the open readingframe as set forth in SEQ ID NO:19.

SEQ ID NO:21 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC4863 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:22 is the amino acid sequence translation of the open readingframe as set forth in SEQ ID NO:21.

SEQ ID NO:23 is an amino acid sequence of a mature TIC3668 protein,mTIC3668.

SEQ ID NO:24 is an amino acid sequence of a mature TIC3669 protein,mTIC3669.

SEQ ID NO:25 is an amino acid sequence of a mature TIC3670 protein,mTIC3670.

SEQ ID NO:26 is an amino acid sequence of a mature TIC4076 protein,mTIC4076.

SEQ ID NO:27 is an amino acid sequence of a mature TIC4078 protein,mTIC4078.

SEQ ID NO:28 is an amino acid sequence of a mature TIC4260 protein,mTIC4260.

SEQ ID NO:29 is an amino acid sequence of a mature TIC4346 protein,mTIC4346.

SEQ ID NO:30 is an amino acid sequence of a mature TIC4826 protein,mTIC4826.

SEQ ID NO:31 is an amino acid sequence of a mature TIC4861 protein,mTIC4891.

SEQ ID NO:32 is a synthetic nucleotide sequence encoding a TIC3668protein designed for expression in plants.

SEQ ID NO:33 is a synthetic nucleotide sequence encoding a matureTIC3668 protein, mTIC3668 designed for expression in plants.

SEQ ID NO:34 is a synthetic nucleotide sequence encoding a TIC3669protein designed for expression in plants.

SEQ ID NO:35 is a synthetic nucleotide sequence encoding a matureTIC3669 protein, mTIC3669 designed for expression in plants.

SEQ ID NO:36 is a synthetic nucleotide sequence encoding a TIC3670protein designed for expression in plants.

SEQ ID NO:37 is a synthetic nucleotide sequence encoding a matureTIC3670 protein, mTIC3670 designed for expression in plants.

SEQ ID NO:38 is a synthetic nucleotide sequence encoding a TIC4076protein designed for expression in plants.

SEQ ID NO:39 is a synthetic nucleotide sequence encoding a matureTIC4076 protein, mTIC4076 designed for expression in plants.

SEQ ID NO:40 is a synthetic nucleotide sequence encoding a TIC4078protein designed for expression in plants.

SEQ ID NO:41 is a synthetic nucleotide sequence encoding a matureTIC4078 protein, mTIC4078 designed for expression in plants.

SEQ ID NO:42 is a synthetic nucleotide sequence encoding a TIC4260protein designed for expression in plants.

SEQ ID NO:43 is a synthetic nucleotide sequence encoding a matureTIC4260 protein, mTIC4260 designed for expression in plants.

SEQ ID NO:44 is a synthetic nucleotide sequence encoding a TIC4346protein designed for expression in plants.

SEQ ID NO:45 is a synthetic nucleotide sequence encoding a matureTIC4346 protein, mTIC4346 designed for expression in plants.

SEQ ID NO:46 is a synthetic nucleotide sequence encoding a TIC4826protein designed for expression in plants.

SEQ ID NO:47 is a synthetic nucleotide sequence encoding a matureTIC4826 protein, mTIC4826 designed for expression in plants.

SEQ ID NO:48 is a synthetic nucleotide sequence encoding a TIC4861protein designed for expression in plants.

SEQ ID NO:49 is a synthetic nucleotide sequence encoding a matureTIC4861 protein (mTIC4861), a mature TIC4862 protein (mTIC4862), and amature TIC4863 protein (mTIC4863) designed for expression in plants.

SEQ ID NO:50 is a synthetic nucleotide sequence encoding a TIC4682protein designed for expression in plants.

SEQ ID NO:51 is a synthetic nucleotide sequence encoding a TIC4863protein designed for expression in plants.

SEQ ID NO:52 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (−) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 1 to36 of SEQ ID NO:1 (TIC3668 forward primer).

SEQ ID NO:53 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (+) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 920to 954 of SEQ ID NO:1 (TIC3668 reverse primer).

SEQ ID NO:54 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (−) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 1 to41 of SEQ ID NO:3 (TIC3669 forward primer).

SEQ ID NO:55 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (+) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 920to 954 of SEQ ID NO:3 (TIC3669 reverse primer).

SEQ ID NO:56 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (−) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 1 to36 of SEQ ID NO:5 (TIC3670 forward primer).

SEQ ID NO:57 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (+) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 920to 954 of SEQ ID NO:5 (TIC3670 reverse primer).

SEQ ID NO:58 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (−) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 1 to41 of SEQ ID NO:7 (TIC4076 forward primer).

SEQ ID NO:59 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (+) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 920to 954 of SEQ ID NO:7 (TIC4076 reverse primer).

SEQ ID NO:60 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (−) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 1 to36 of SEQ ID NO:9 (TIC4078 forward primer).

SEQ ID NO:61 is a nucleotide sequence representing a syntheticoligonucleotide for hybridizing to the (+) strand of a DNA encoding aprotein disclosed in this application and corresponds to positions 920to 954 of SEQ ID NO:9 (TIC4078 reverse primer).

SEQ ID NO:62 is a recombinant polynucleotide sequence obtained from aBrevibacillus laterosporus species encoding a TIC2462 protein from anopen reading frame at nucleotide position 1-951 and a translationtermination codon.

SEQ ID NO:63 is the amino acid sequence translation of the open readingframe as set forth in SEQ ID NO:62.

SEQ ID NO:64 is a synthetic nucleotide sequence encoding a matureTIC3668 protein, mTIC3668 for expression in bacteria.

SEQ ID NO:65 is a synthetic nucleotide sequence encoding a matureTIC3669 protein, mTIC3669 for expression in bacteria.

SEQ ID NO:66 is a synthetic nucleotide sequence encoding a matureTIC3670 protein, mTIC3670 for expression in bacteria.

SEQ ID NO:67 is a synthetic nucleotide sequence encoding a matureTIC4076 protein, mTIC4076 for expression in bacteria.

SEQ ID NO:68 is a synthetic nucleotide sequence encoding a matureTIC4078 protein, mTIC4078 for expression in bacteria.

SEQ ID NO:69 is a synthetic nucleotide sequence encoding a matureTIC4260 protein, mTIC4260 for expression in bacteria.

SEQ ID NO:70 is a synthetic nucleotide sequence encoding a matureTIC4346 protein, mTIC4346 for expression in bacteria.

SEQ ID NO:71 is a synthetic nucleotide sequence encoding a matureTIC4826 protein, mTIC4826 for expression in bacteria.

SEQ ID NO:72 is a synthetic nucleotide sequence encoding a matureTIC4861 (mTIC4861), TIC4862 (mTIC4862), and TIC4863 (mTIC4863)proteinfor expression in bacteria.

DETAILED DESCRIPTION OF THE INVENTION

The problem in the art of agricultural pest control can be characterizedas a need for new toxin proteins that are efficacious against targetpests, exhibit broad spectrum toxicity against target pest species, arecapable of being expressed in plants without causing undesirableagronomic issues, and provide an alternative mode of action compared tocurrent toxins that are used commercially in plants. Novel insecticidalproteins exemplified by TIC3668 are disclosed herein, and address eachof these needs, particularly against a broad spectrum of Coleopteran andLepidopteran insect pests, and more particularly against corn rootwormpest species.

Reference in this application to “TIC3668”, “TIC3668 protein”, “TIC3668protein toxins”, “TIC3668 toxin proteins”, “TIC3668-related toxins”,“TIC3668-related protein toxin class or family”, “TIC3668-related toxinproteins”, “TIC3668-type proteins”, “TIC3668-like proteins,“TIC3668-related toxin polypeptides”, “TIC3668-related pesticidalproteins”, or “TIC3668-type insect inhibitory polypeptide” and the like,refer to any novel insect inhibitory protein that comprises, thatconsists of, that is substantially homologous to, that is similar to, orthat is derived from any insect inhibitory polypeptide sequence ofTIC3668 (SEQ ID NO:2) and insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests andLepidopteran pests, including any protein exhibiting insect inhibitoryactivity if alignment of such protein with TIC3668 (SEQ ID NO:2),TIC3669 (SEQ ID NO:4), TIC3670 (SEQ ID NO:6), TIC4076 (SEQ ID NO:8),TIC4078 (SEQ ID NO:10), TIC4346 (SEQ ID NO:14), TIC4826 (SEQ ID NO:16),TIC4861 (SEQ ID NO:18), TIC4862 (SEQ ID NO:20), and TIC4863 (SEQ IDNO:22), results in amino acid sequence identity of any fractionpercentage from about 35% to about 100% percent. The TIC3668-typeprotein toxins disclosed in this application include TIC3668, TIC3669,TIC3670, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862, TIC4863,and the collage TIC4260 protein (SEQ ID NO:12). The TIC3668-type proteinclass is intended to include the precursor forms as well as the maturelength forms of the proteins.

The term “segment” or “fragment” is used in this application to describeconsecutive amino acid or nucleic acid sequences that are shorter thanthe complete amino acid or nucleic acid sequence describing aTIC3668-type protein. A segment or fragment exhibiting insect inhibitoryactivity is also disclosed in this application if alignment of suchsegment or fragment, with the corresponding section of the TIC3668-typeprotein set forth in SEQ ID NO:2, results in amino acid sequenceidentity of any fraction percentage from about 35 to about 100 percentbetween the segment or fragment and the corresponding section of theTIC3668-type protein.

Reference in this application to the terms “active” or “activity”,“pesticidal activity” or “insecticidal activity”, “insect inhibitory” or“insecticidal” refer to efficacy of a toxic agent, such as a proteintoxin, in inhibiting (inhibiting growth, feeding, fecundity, orviability), suppressing (suppressing growth, feeding, fecundity, orviability), controlling (controlling the pest infestation, controllingthe pest feeding activities on a particular crop containing an effectiveamount of the TIC3668-type protein) or killing (causing the morbidity,mortality, or reduced fecundity of) a pest. These terms are intended toinclude the result of providing a pesticidally effective amount of atoxic protein to a pest where the exposure of the pest to the toxicprotein results in morbidity, mortality, reduced fecundity, or stunting.These terms also include repulsion of the pest from the plant, a tissueof the plant, a plant part, seed, plant cells, or from the particulargeographic location where the plant may be growing, as a result ofproviding a pesticidally effective amount of the toxic protein in or onthe plant. In general, pesticidal activity refers to the ability of atoxic protein to be effective in inhibiting the growth, development,viability, feeding behavior, mating behavior, fecundity, or anymeasurable decrease in the adverse effects caused by an insect feedingon this protein, protein fragment, protein segment or polynucleotide ofa particular target pest, including but not limited to insects of theorder Lepidoptera or Coleoptera. The toxic protein can be produced bythe plant or can be applied to the plant or to the environment withinthe location where the plant is located. The terms “bioactivity”,“effective”, “efficacious” or variations thereof are also termsinterchangeably utilized in this application to describe the effects ofproteins of the present invention on target insect pests.

A pesticidally effective amount of a toxic agent, when provided in thediet of a target pest, exhibits pesticidal activity when the toxic agentcontacts the pest. A toxic agent can be a pesticidal protein or one ormore chemical agents known in the art. Insecticidal chemical agents andinsecticidal protein agents can be used alone or in combinations witheach other. Chemical agents include but are not limited to dsRNAmolecules targeting specific genes for suppression in a target pest,organochlorides, organophosphates, carbamates, pyrethroids,neonicotinoids, and ryanoids. Insecticidal protein agents include theprotein toxins set forth in this application, as well as otherproteinaceous toxic agents including those that target Lepidopteran andColeopteran, as well as protein toxins that are used to control otherplant pests such as Cry proteins available in the art for use incontrolling Hemipteran and Homopteran species.

It is intended that reference to a pest, particularly a pest of a cropplant, means insect pests of crop plants, particularly those that arecontrolled by the TIC3668-related protein toxin class. However,reference to a pest can also include Hemipteran and Homopteran insectpests of plants, as well as nematodes and fungi when toxic agentstargeting these pests are co-localized or present together with one ormore proteins of the TIC3668-related protein toxin class.

The individual proteins which comprise the TIC3668-related protein classare related by common function and exhibit insecticidal activity towardsinsect pests from the Coleoptera and Lepidoptera insect species,including adults, pupae, larvae, and neonates. The insects of the orderLepidoptera include, but are not limited to, armyworms, cutworms,loopers, and heliothines in the Family Noctuidae, e.g., fall armyworm(Spodoptera frugiperda), beet armyworm (Spodoptera exigua), berthaarmyworm (Mamestra configurata), black cutworm (Agrotis ipsilon),cabbage looper (Trichoplusia ni), soybean looper (Pseudoplusiaincludens), velvetbean caterpillar (Anticarsia gemmatalis), greencloverworm (Hypena scabra), tobacco budworm (Heliothis virescens),granulate cutworm (Agrotis subterranea), armyworm (Pseudaletiaunipuncta), western cutworm (Agrotis orthogonia); borers, casebearers,webworms, coneworms, cabbageworms and skeletonizers from the FamilyPyralidae, e.g., European corn borer (Ostrinia nubilalis), navelorangeworm (Amyelois transitella), corn root webworm (Crambuscaliginosellus), sod webworm (Herpetogramma licarsisalis), sunflowermoth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpuslignosellus); leafrollers, budworms, seed worms, and fruit worms in theFamily Tortricidae, e.g., codling moth (Cydia pomonella), grape berrymoth (Endopiza viteana), oriental fruit moth (Grapholita molesta),sunflower bud moth (Suleima helianthana); and many other economicallyimportant Lepidoptera, e.g., diamondback moth (Plutella xylostella),pink bollworm (Pectinophora gossypiella) and gypsy moth (Lymantriadispar). Other insect pests of order Lepidoptera include, e.g., Alabamaargillacea (cotton leaf worm), Archips argyrospila (fruit tree leafroller), Archips rosana (European leafroller) and other Archips species,Chilo suppressalis (Asiatic rice borer, or rice stem borer),Cnaphalocrocis medinalis (rice leaf roller), Crambus caliginosellus(corn root webworm), Crambus teterrellus (bluegrass webworm), Diatraeagrandiosella (southwestern corn borer), Diatraea saccharalis (surgarcaneborer), Earias insulana (spiny bollworm), Earias vittella (spottedbollworm), Helicoverpa armigera (American bollworm), Helicoverpa zea(corn earworm or cotton bollworm), Heliothis virescens (tobaccobudworm), Herpetogramma licarsisalis (sod webworm), Lobesia botrana(European grape vine moth), Phyllocnistis citrella (citrus leafminer),Pieris brassicae (large white butterfly), Pieris rapae (importedcabbageworm, or small white butterfly), Plutella xylostella (diamondbackmoth), Spodoptera exigua (beet armyworm), Spodoptera litura (tobaccocutworm, cluster caterpillar), and Tuta absoluta (tomato leafminer). Theinsects of the order Coleoptera include, but are not limited to,Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp, particularly when the pest is Diabrotica virgiferavirgifera (Western Corn Rootworm, WCR), Diabrotica barberi (NorthernCorn Rootworm, NCR), Diabrotica virgifera zeae (Mexican Corn Rootworm,MCR), Diabrotica balteata (Brazilian Corn Rootworm (BZR), Diabroticaundecimpunctata howardii (Southern Corn Rootworm, SCR) and a BrazilianCorn Rootworm complex (BCR) consisting of Diabrotica viridula andDiabrotica speciosa).

Reference in this application to an “isolated DNA molecule”, “isolatedpolynucleotide molecule”, or an equivalent term or phrase, is intendedto mean that the DNA molecule is one that is present alone or incombination with other compositions, but not within its naturalenvironment. For example, nucleic acid elements such as a codingsequence, intron sequence, untranslated leader sequence, promotersequence, transcriptional termination sequence, and the like, that arenaturally found within the DNA of the genome of an organism are notconsidered to be “isolated” so long as the element is within the genomeof the organism and at the location within the genome in which it isnaturally found. However, each of these elements, and subparts of theseelements, would be “isolated” within the scope of this disclosure solong as the element is not within the genome of the organism and at thelocation within the genome in which it is naturally found. Similarly, anucleotide sequence encoding a insecticidal protein or any naturallyoccurring insecticidal variant of that protein would be an isolatednucleotide sequence so long as the nucleotide sequence was not withinthe DNA of the bacterium from which the sequence encoding the protein isnaturally found. A synthetic nucleotide sequence encoding the amino acidsequence of the naturally occurring insecticidal protein would beconsidered to be isolated for the purposes of this disclosure. For thepurposes of this disclosure, any transgenic nucleotide sequence, i.e.,the nucleotide sequence of the DNA inserted into the genome of the cellsof a plant or bacterium, or present in an extrachromosomal vector wouldbe considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

As described further in this application, an open reading frame (ORF)(SEQ ID NO:1) encoding TIC3668 (SEQ ID NO:2) was discovered in DNAobtained from Brevibacillus laterosporus strain EG5552. Other bacterialgenomes were then screened for sequences encoding TIC3668-relatedprotein. Several other open reading frames were identified in theseother bacterial genomes encoding amino acid sequences resembling theEG5552 TIC3668 protein, including the TIC3668-like proteins TIC3669which was discovered in DNA obtained from Brevibacillus laterosporusstrain EG5551 (SEQ ID NO:3 encoding SEQ ID NO:4), TIC3670 which wasdiscovered in DNA obtained from Brevibacillus laterosporus strain EG5553(SEQ ID NO:5 encoding SEQ ID NO:6), TIC4076 which was discovered in DNAobtained from Brevibacillus laterosporus strain ATCC6456 (SEQ ID NO:7encoding SEQ ID NO:8), TIC4078 which was discovered in DNA obtained fromBrevibacillus laterosporus strain EG4227 (SEQ ID NO:9 encoding SEQ IDNO:10), TIC4346 which was discovered in DNA obtained from Brevibacilluslaterosporus strain EG5551 (SEQ ID NO:13 encoding SEQ ID NO:14), TIC4826which was discovered in DNA obtained from Brevibacillus laterosporusstrain AG0021D10 (SEQ ID NO:15 encoding SEQ ID NO:16). TIC4861 (SEQ IDNO:17 encoding SEQ ID NO:18), TIC4862 (SEQ ID NO:19 encoding SEQ IDNO:20) and TIC4863 (SEQ :ID NO:21 encoding SEQ ID NO:22) which werediscovered in DNA obtained from Brevibacillus laterosporus strainEG4227. One additional TIC3668-like protein, TIC4260 (SEQ ID NO:11encoding SEQ ID NO:12), was created by combining the naturally occurringamino acid sequence variation from five different native TIC3668-likeproteins to create a collage protein.

The respective coding sequences were cloned and expressed in microbialhost cells to produce recombinant proteins for use in insect bioassays.As described further in this application, it is shown that theseproteins exhibit bioactivity against Diabrotica species, includingWestern Corn Rootworm (WCR, Diabrotica virgifera virgifera), WesternEuropean Corn Borer (ECB, Ostrinia nubialis), Southwestern Corn Borer(SWC, Diatraea grandiosella), and Soybean Looper (SBL, Chrysodeixisincludens).

A surprising feature of the TIC3668-type proteins is the presence of aN-terminal amino acid segment corresponding to amino acid position 1 to23 for TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346,TIC4826, TIC4863; 1 to 12 for TIC4861; and 1 to 21 for TIC4862. Each ofthese N-terminal amino acid segments may be omitted from the respectiveprotein and the polynucleotide sequence encoding the respective segmentmay also be omitted. When expressed in planta, omission of theserespective segments surprisingly resulted in an increase of insecticidalactivity against corn rootworm species compared to expression of thefull-length protein toxin containing the omitted segment. Protein toxinsegments lacking the N-terminal amino acid segments referred to aboveare referred to herein as “mature TIC3668-type toxin proteins”. Ingeneral, reference to the mature version of a TIC3668-type protein isannotated herein with the letter “m” preceding the name of the toxin todifferentiate reference to the mature sequence from the full lengthnative sequence. For example, the mature version of the amino acidsequence for TIC3668 (SEQ ID NO: 2) is mTIC3668 (SEQ ID NO:23). Themature versions for TIC3669 (SEQ ID NO:4), TIC3670 (SEQ ID NO:6),TIC4076 (SEQ ID NO:8), TIC4078 (SEQ ID NO:10), TIC4260 (SEQ ID NO:12),TIC4346 (SEQ ID NO:14) and TIC4826 (SEQ ID NO:16) are mTIC3669 (SEQ IDNO:24), mTIC3670 (SEQ ID NO:25), mTIC4076 (SEQ ID NO:26), mTIC4078 (SEQID NO:27), mTIC4260 (SEQ ID NO:28), mTIC4346 (SEQ ID NO:29) and mTIC4826(SEQ ID NO:30), respectively. The full-length proteins TIC4861 (SEQ IDNO:18), TIC4862 (SEQ ID NO:20) and TIC4863 (SEQ ID NO:22) are sequencelength variants of each other and differ only in the length of theirN-terminal amino acid segment. Removal of the N-terminal amino acidsegment in TIC4861, TIC4862, and TIC4863 creates an identical matureamino acid sequence for mTIC4861, mTIC4862, and mTIC4863. Thus, theamino acid sequences for mTIC4861, mTIC4862, and mTIC4863 are encoded bythe same polynucleotide sequence (mTIC4861, SEQ ID NO:31). The matureTIC3668-like protein sequences are encoded by SEQ ID NO:64 (encodingmTIC3668), SEQ ID NO:65 (encoding mTIC3669), SEQ ID NO:66 (encodingmTIC3670), SEQ ID NO:67 (encoding mTIC4076), SEQ ID NO:68 (encodingmTIC4078), SEQ ID NO:69 (encoding mTIC4260), SEQ ID NO:70 (encodingmTIC4346), SEQ ID NO:71 (encoding mTIC4826), and SEQ ID NO.72 (encodingmTIC4861, mTIC4862, and mTIC4863) for expression in bacterial hosts.

Additional members to the TIC3668-type family can be created by usingthe naturally occurring amino acid variations from some or all familymembers to create novel proteins of a higher level of amino acidsequence diversity and with novel properties. Variants of theTIC3668-type protein toxin class were produced by aligning the aminoacid sequences of TIC3668-type family members and combining differencesat the amino acid sequence level into a novel amino acid sequence andmaking appropriate changes to the polynucleotides encoding thesevariants. One such example is TIC4260. SEQ ID NO:11 is thepolynucleotide sequence encoding the TIC4260 protein (SEQ ID NO:12). Themature protein (mTIC4260, SEQ ID NO:28) is encoded by the polynucleotidesequence of SEQ ID NO:43.

Fragments of the TIC3668-type protein toxins can be truncated formswherein one or more amino acids are deleted from the N-terminal end,C-terminal end, the middle of the protein, or combinations thereof withinsect inhibitory activity. These fragments can be naturally occurringor synthetic variants of TIC3668, TIC3669, TIC3670, TIC4260, TIC4076,TIC4078, TIC4346, TIC4826, TIC4861, TIC4862 or TIC4863, but shouldretain or improve the insect inhibitory activity of TIC3668, TIC3669,TIC3670, TIC4260, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862or TIC4863. Truncated N-terminal or C-terminal deletion variantsinclude, but are not limited to, TIC3668, TIC3669, TIC3670, TIC4260,TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862 or TIC4863 proteinsthat lack amino acid residues from either the N-terminus and/or theC-terminus. For example, N-terminal amino acid residues 1 to 23 of aTIC3668 protein can be deleted resulting in a toxin protein having aminoacids 24-317 of SEQ ID NO:2. Removing 10 or 20 amino acids from theC-terminal amino acid end of a TIC3668 protein resulted in a loss ofinsecticidal activity, while removing a single amino acid did not affectactivity.

Proteins of the TIC3668-type protein class, and proteins that resemblethe proteins of the TIC3668-type protein class, can be identified bycomparison to each other using various computer based algorithms knownin the art (see Tables 1 and 2). Amino acid sequence identities reportedherein are a result of a Clustal W alignment using these defaultparameters: Weight matrix: blosum, Gap opening penalty: 10.0, Gapextension penalty: 0.05, Hydrophilic gaps: On, Hydrophilic residues:GPSNDQERK, Residue-specific gap penalties: On (Thompson, et al. (1994)Nucleic Acids Research, 22:4673-4680). Percent amino acid identity isfurther calculated by the product of 100% multiplied by (amino acididentities/length of subject protein). Other alignment algorithms arealso available in the art and provide results similar to those obtainedusing a Clustal W alignment.

It is intended that a protein exhibiting insect inhibitory activityagainst a Lepidopteran insect species is a member of the TIC3668-typeprotein toxin class if the protein is used in a query, e.g., in aClustal W alignment, and at least one of the proteins of the presentinvention as set forth as mTIC4260 is identified as hits in suchalignment in which the query protein exhibits at least about 85% toabout 100% amino acid sequence identity along the length of the queryprotein, that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 100%, or any fraction percentage in this range; orat least one of the proteins of the present invention as set forth asmTIC3668 is identified as hits in such alignment in which the queryprotein exhibits at least about 89% to about 100% amino acid sequenceidentity along the length of the query protein, that is 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any fraction percentagein this range; or at least one of the proteins of the present inventionas set forth as mTIC3669 and/or mTIC3670 are identified as hits in suchalignment in which the query protein exhibits at least about 90% toabout 100% amino acid sequence identity along the length of the queryprotein, that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,or any fraction percentage in this range; or at least one of theproteins of the present invention as set forth as mTIC4826 is identifiedas a hit in such alignment in which the query protein exhibits at leastabout 91% to about 100% amino acid sequence identity along the length ofthe query protein, that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, or any fraction percentage in this range.

It is intended that a protein exhibiting insect inhibitory activityagainst a Coleopteran insect species is a member of the TIC3668-typeprotein toxin class if the protein is used in a query, e.g., in aClustal W alignment, and at least one of the proteins of the presentinvention as set forth as mTIC3668, mTIC3669, mTIC3670, mTIC4076,mTIC4078, mTIC4260, mTIC4346, mTIC4826, mTIC4861, mTIC4862, and mTIC4863are identified as hits in such alignment in which the query proteinexhibits at least about 35% to about 100% amino acid identity along thelength of the query protein that is about 35%, 40%, 50%, 60%, 70%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any fractionpercentage in this range.

Exemplary proteins of the TIC3668-type protein toxin class were alignedwith each other using a Clustal W algorithm. A pair-wise matrix ofpercent amino acid sequence identities for each pair of the full-lengthproteins was created, as reported in Table 1. A pair-wise matrix ofpercent amino acid sequence identities for each pair of themature-length proteins was created, as reported in Table 2.

TABLE 1 Pair-wise matrix display of exemplary full-length proteins N SEQID NO: M 2 6 4 8 14 18 20 22 16 10 12 2 TIC3668 — 99.4 97.8 96.2 97.293.1 95.6 96.5 97.2 94.3 96.2 (315) (310) (305) (308) (295) (303) (306)(308) (299) (305) 6 TIC3670 99.4 — 98.4 96.8 97.2 93.7 96.2 97.2 97.8 9595.6 (315) (312) (307) (308) (297) (305) (308) (310) (301) (303) 4TIC3669 97.8 98.4 — 96.8 96.8 93.4 96.2 97.2 97.5 94.6 95.3 (310) (312)(307) (307) (296) (305) (308) (309) (300) (302) 8 TIC4076 96.2 96.8 96.8— 98.4 94.3 97.2 98.1 98.1 96.2 93.4 (305) (307) (307) (312) (299) (308)(311) (311) (305) (296) 14 TIC4346 97.2 97.2 96.8 98.4 — 94.3 97.2 98.198.7 96.2 93.7 (308) (308) (307) (312) (299) (308) (311) (313) (305)(297) 18 TIC4861 96.4 97.1 96.7 97.7 97.7 — 99.7 99.7 98.4 95.4 92.5(295) (297) (296) (299) (299) (305) (305) (301) (292) (283) 20 TIC486296.2 96.8 96.8 97.8 97.8 96.8 — 99.7 98.4 95.2 92.4 (303) (305) (305)(308) (308) (305) (314) (310) (300) (291) 22 TIC4863 96.5 97.2 97.2 98.198.1 96.2 99.1 — 98.7 95.6 92.7 (306) (308) (308) (311) (311) (305)(314) (313) (303) (294) 16 TIC4826 97.2 97.8 97.5 98.1 98.7 95 97.8 98.7— 95.9 93.4 (308) (310) (309) (311) (313) (301) (310) (313) (304) (296)10 TIC4078 94.3 95 94.6 96.2 96.2 92.1 94.6 95.6 95.9 — 96.2 (299) (301)(300) (305) (305) (292) (300) (303) (304) (305) 12 TIC4260 96.2 95.695.3 93.4 93.7 89.3 91.8 92.7 93.4 96.2 — (305) (303) (302) (296) (297)(283) (291) (294) (296) (305) Table Description: Clustal W alignmentbetween (X) versus (Y) are reported in a pair-wise matrix. Columns under(N) refer to SEQ ID NO. Column (M) refers to protein name (TIC#). Thepercent amino acid identity between all pairs is calculated and isrepresented by the first number in each box. The second number (inparentheses) in each box represents the number of identical amino acidsbetween the pair.

TABLE 2 Pair-wise matrix display of exemplary mature proteins N SEQ IDNO: M 26 29 30 31 23 25 24 27 28 26 mTIC4076 — 98.3 98 98 96.3 96.9 96.696.3 93.2 (290) (289) (289) (284) (286) (285) (284) (275) 29 mTIC434698.3 — 98.6 98 97.3 97.3 96.6 96.3 93.6 (290) (291) (289) (287) (287)(285) (284) (276) 30 mTIC4826 98 98.6 — 98.6 97.3 98 97.3 95.9 93.2(289) (291) (291) (287) (289) (287) (283) (275) 31 mTIC4861 98 98 98.6 —96.6 97.3 96.9 95.6 92.5 mTIC4862 (289) (289) (291) (285) (287) (286)(282) (273) mTIC4863 23 mTIC3668 96.3 97.3 97.3 96.6 — 99.3 98 93.9 95.9(284) (287) (287) (285) (293) (289) (277) (283) 25 mTIC3670 96.9 97.3 9897.3 99.3 — 98.6 94.6 95.3 (286) (287) (289) (287) (293) (291) (279)(281) 24 mTIC3669 96.6 96.6 97.3 96.9 98 98.6 — 94.6 95.3 (285) (285)(287) (286) (289) (291) (279) (281) 27 mTIC4078 96.3 96.3 95.9 95.6 93.994.6 94.6 — 95.9 (284) (284) (283) (282) (277) (279) (279) (283) 28mTIC4260 93.2 93.6 93.2 92.5 95.9 95.3 95.3 95.9 — (275) (276) (275)(273) (283) (281) (281) (283) Table Description: Clustal W alignmentbetween (X) versus (Y) are reported in a pair-wise matrix. Columns under(N) refer to SEQ ID NO. Column (M) refers to protein name (TIC#). Thepercent amino acid identity between all pairs is calculated and isrepresented by the first number in each box. The second number (inparentheses) in each box represents the number of identical amino acidsbetween the pair.

The full-length and mature proteins of the TIC3668-type protein toxinclass can also be related by primary structure (conserved amino acidmotifs), by length (about 295 amino acids for the mature proteins andabout 317 amino acids for the full-length proteins) and by othercharacteristics. The full-length proteins from the present inventionhave a measured mass of about 35k-Daltons when run on protein gels underdenaturing conditions, and the mature proteins have a measured mass ofabout 32 kDa. Characteristics of the full-length and mature forms of theTIC3668-type protein toxin class are reported in Tables 3 and 4.

TABLE 3 Characteristics of Full-length Protein No. of No. of MolecularAmino Strongly Strongly No. of No. of Weight Acid Isoelectric Charge atBasic (−) Acidic Hydrophobic Polar Protein (in Daltons) Length Point PH7.0 Amino Acids Amino Acids Amino Acids Amino Acids TIC3668 34770.96 3179.049 5.229 34 29 95 111 TIC3669 34769.91 317 8.898 4.231 34 30 95 111TIC3670 34788.89 320 8.898 4.231 34 30 93 112 TIC4076 34652.83 317 8.7213.232 32 29 95 112 TIC4078 34676.86 317 8.936 4.397 32 28 96 110 TIC426034743.98 317 9.077 5.395 33 28 96 109 TIC4826 34734.97 317 8.899 4.23133 29 95 111 TIC4861 33448.24 306 8.439 2.233 31 29 87 110 TIC486234392.43 315 8.439 2.233 31 29 94 112 TIC4863 34648.77 317 8.899 4.23133 29 94 112 TIC4346 34717.95 317 8.437 2.235 32 30 97 109

TABLE 4 Characteristics of Mature Protein No. of No. of No. of MolecularAmino Iso- Strongly Strongly Hydro- No. of Weight Acid electric ChargeBasic Acidic phobic Polar Protein (in Daltons) Length Point at PH 7.0Amino Acids Amino Acids Amino Acids Amino Acids mTIC3668 32317.06 2958.722 3.064 32 29 83 104 mTIC3669 32303.95 295 8.436 2.067 32 30 82 105mTIC3670 32334.99 295 8.436 2.067 32 30 81 105 mTIC4076 32186.87 2958.000 1.068 30 29 82 106 mTIC4078 32222.96 295 8.466 2.233 30 28 84 103mTIC4260 32290.07 295 8.747 3.230 31 28 84 102 mTIC4826 32269.01 2958.436 2.066 31 29 82 105 mTIC4861 32182.81 295 8.436 2.066 31 29 81 106mTIC4862 mTIC4863 mTIC4346 32251.99 295 7.092 0.071 30 30 84 103

The proteins of the disclosed TIC3668-type protein toxin class representa new class of insecticidal proteins. With reference to Table 5, all ofthe numbers above the diagonal line corresponding to 100% identity,represent the number of amino acid differences between the correspondingproteins being compared at the intersection of that particular row andcolumn. The numbers below the diagonal line corresponding to 100%identity represent the percent identity of the corresponding proteinsbeing compared at the intersection of that particular row and column.The mature length members of this protein class exhibit no greater than90.54% amino acid identity to any other insecticidal protein known inthe art, as demonstrated in the alignment provided in Table 5. Theinsecticidal protein exhibiting the nearest identity to any of themature length proteins of the present invention is SEQ ID NO:50 in U.S.Patent Application Publication number 20110030093 (AXMI-209) with 90.5%sequence identity to mTIC4076, mTIC4346, mTIC4826, and mTIC4863. Thisdisclosure only teaches activity against Lepidoptera, while exemplaryproteins of the present invention demonstrate activity againstColeoptera. HOUDD3_BRELA, F7TVP6_BRELA, and U4WSU1_BRELA are unannotatedprotein sequences predicted from the open reading frame in genomesequences reported as having been obtained from B. laterosporous. Noinsecticidal activity is reported for these proteins.

TABLE 5 Alignment of Mature Length TIC3886 Proteins to Prior ArtProteins 1 2 3 4 5 6 7 8 9 10 11 12 13 mTIC3668 1 100 6 2 11 18 12 8 810 36 32 32 33 mTIC3669 2 98.0 100 4 10 16 14 10 8 9 35 32 32 32mTIC3670 3 99.3 98.6 100 9 16 14 8 6 8 34 30 30 31 mTIC4076 4 96.3 96.697.0 100 11 20 5 6 6 30 28 30 29 mTIC4078 5 93.9 94.6 94.6 96.3 100 1211 12 13 37 34 36 34 mTIC4260 6 95.9 95.3 95.3 93.2 95.9 100 19 20 22 4844 44 43 mTIC4346 7 97.3 96.6 97.3 98.3 96.3 93.6 100 4 6 30 26 28 29mTIC4826 8 97.3 97.3 98.0 98.0 95.9 93.2 98.6 100 4 30 24 24 25 mTIC48639 96.6 97.0 97.3 98.0 95.6 92.5 98.0 98.6 100 30 28 28 27 AXMI_209 1088.6 89.0 89.3 90.5 88.3 84.9 90.5 90.5 90.4 100 6 8 7 H0UDD3_ 11 89.989.9 90.5 91.2 89.3 86.1 91.8 92.4 91.2 98.1 100 2 3 BRELA F7TVP6_ 1289.9 89.9 90.5 90.5 88.6 86.1 91.2 92.4 91.2 97.5 99.4 100 3 BRELAU4WSU1_ 13 89.6 89.9 90.2 90.9 89.3 86.4 90.9 92.1 91.5 97.8 99.1 99.1100 BRELA

The TIC3668 proteins disclosed in this application exhibit activity indiet bioassays against Coleoptera, including WCR. In some casesLepidopteran activity is also observed.

As described further in the Examples of this application, polynucleotidesequences encoding TIC3668 toxin proteins were designed for use inplants. Exemplary polynucleotides that were designed for expression inplants and encode the full-length of the insect inhibitory TIC3668,TIC3669, TIC3670, TIC4260, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861,TIC4862, and TIC4863 proteins are set forth in SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:51.Exemplary polynucleotides that were designed for expression in plantsand encode a mature form of the insect inhibitory mTIC3668, mTIC3669,mTIC3670, mTIC4260, mTIC4076, mTIC4078, mTIC4346, mTIC4826, mTIC4861,mTIC4862, and mTIC4863 proteins are set forth in SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO: 43, SEQ IDNO:45, SEQ ID NO:47, and SEQ ID NO:49.

Expression cassettes and vectors containing these polynucleotidesequences were constructed and introduced into corn plant cells inaccordance with transformation methods and techniques known in the art.Transformed cells were regenerated into transformed plants that wereobserved to be expressing TIC3668 toxin proteins. To test pesticidalactivity, bioassays were performed in the presence of Lepidopteran orColeopteran pest larvae using plant leaf disks obtained from thetransformed plants.

The insect inhibitory activity of exemplary members of the TIC3668-typeprotein toxin class is described in more detail in the Examples. Theexemplary proteins are related by common function and exhibitinsecticidal activity towards Coleoptera and Lepidoptera insect species,including adults, pupae, larvae and neonates.

Recombinant polynucleotide compositions that encode TIC3668-typeproteins are contemplated. For example, TIC3668-type proteins can beexpressed with recombinant DNA constructs in which a polynucleotidemolecule with an ORF encoding the protein is operably linked to geneticexpression elements such as a promoter and any other regulatory elementnecessary for expression in the system for which the construct isintended. Non-limiting examples include a plant-functional promoteroperably linked to the TIC3668-type protein encoding sequences forexpression of the protein in plants or a Bt-functional promoter operablylinked to a TIC3668-type protein encoding sequence for expression of theprotein in a Bt bacterium or other Bacillus species. Other elements canbe operably linked to the TIC3668-type protein encoding sequencesincluding, but not limited to, enhancers, introns, untranslated leaders,encoded protein immobilization tags (HIS-tag), translocation peptides(i.e., plastid transit peptides, signal peptides), polypeptide sequencesfor post-translational modifying enzymes, ribosomal binding sites, andRNAi target sites. Exemplary recombinant polynucleotide moleculesprovided herewith include, but are not limited to, a heterologouspromoter operably linked to a polynucleotide such as SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21 that encodesthe respective polypeptides or proteins having the amino acid sequenceas set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, and SEQ ID NO:22. The codons of a recombinant polynucleotidemolecule encoding for proteins disclosed herein can be substituted bysynonymous codons (known in the art as a silent substitution).Non-limiting examples for modified polynucleotides encoding any of theTIC3668-type proteins disclosed in this application are set forth in SEQID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQID NO:51 for the full-length protein sequences and SEQ ID NOs:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, and SEQ ID NO:49 for the mature protein sequences.

A recombinant DNA construct comprising TIC3668-type protein encodingsequences can further comprise a region of DNA that encodes for one ormore insect inhibitory agents which can be configured to concomitantlyexpress or co-express with a DNA sequence encoding a TIC3668-typeprotein, a protein different from a TIC3668-type protein, an insectinhibitory dsRNA molecule, or an ancillary protein. Ancillary proteinsinclude, but are not limited to, co-factors, enzymes, binding-partners,or other agents that function to aid in the effectiveness of an insectinhibitory agent, for example, by aiding its expression, influencing itsstability in plants, optimizing free energy for oligomerization,augmenting its toxicity, and increasing its spectrum of activity. Anancillary protein may facilitate the uptake of one or more insectinhibitory agents, for example, or potentiate the toxic effects of thetoxic agent.

A recombinant DNA construct can be assembled so that all proteins ordsRNA molecules are expressed from one promoter or each protein or dsRNAmolecules is under separate promoter control or some combinationthereof. The proteins of this invention can be expressed from amulti-gene expression system in which one or more proteins of theTIC3668-proteins are expressed from a common nucleotide segment whichalso contains other open reading frames and promoters, depending on thetype of expression system selected. For example, a bacterial multi-geneexpression system can utilize a single promoter to drive expression ofmultiply-linked/tandem open reading frames from within a single operon(i.e., polycistronic expression). In another example, a plant multi-geneexpression system can utilize multiply-unlinked expression cassetteseach expressing a different protein or other agent such as one or moredsRNA molecules

Recombinant polynucleotides or recombinant DNA constructs comprising aTIC3668-type protein encoding sequence can be delivered to host cells byvectors, e.g., a plasmid, baculovirus, synthetic chromosome, virion,cosmid, phagemid, phage, or viral vector. Such vectors can be used toachieve stable or transient expression of a TIC3668-type proteinencoding sequence in a host cell, or subsequent expression of theencoded polypeptide. An exogenous recombinant polynucleotide orrecombinant DNA construct that comprises a TIC3668-type protein encodingsequence and that is introduced into a host cell is referred herein as a“transgene”.

Transgenic bacteria, transgenic plant cells, transgenic plants, andtransgenic plant parts that contain a recombinant polynucleotide thatexpresses any one or more of the TIC3668-type protein encoding sequencesare provided herein. The term “bacterial cell” or “bacterium” caninclude, but is not limited to, an Agrobacterium, a Bacillus, anEscherichia, a Salmonella, a Pseudomonas, or a Rhizobium cell. The term“plant cell” or “plant” can include but is not limited to amonocotyledon, dicotyledon, alfalfa, banana, barley, bean, broccoli,cabbage, brassica, carrot, cassava, castor, cauliflower, celery,chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover,cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax,garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut,oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper,pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish,rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southernpine, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cellor plant. In certain embodiments, transgenic plants and transgenic plantparts regenerated from a transgenic plant cell are provided. In certainembodiments, the transgenic plants can be obtained from a transgenicseed, by cutting, snapping, grinding or otherwise disassociating thepart from the plant. In certain embodiments, the plant part can be aseed, a boll, a leaf, a flower, a stem, a root, or any portion thereof,or a non-regenerable portion of a transgenic plant part. As used in thiscontext, a “non-regenerable” portion of a transgenic plant part is aportion that can not be induced to form a whole plant or that can not beinduced to form a whole plant that is capable of sexual and/or asexualreproduction. In certain embodiments, a non-regenerable portion of aplant part is a portion of a transgenic seed, boll, leaf, flower, stem,or root.

Methods of making transgenic plants that comprise insect, Coleoptera- orLepidoptera-inhibitory amounts of a TIC3668-type protein are provided.Such plants can be made by introducing a recombinant polynucleotide thatencodes any of the TIC3668-type proteins provided in this applicationinto a plant cell, and selecting a plant derived from said plant cellthat expresses an insect, Coleoptera- or Lepidoptera-inhibitory amountof the TIC3668-type proteins. Plants can be derived from the plant cellsby regeneration, seed, pollen, or meristem transformation techniques.Methods for transforming plants are known in the art.

Processed plant products, wherein the processed product comprises adetectable amount of a TIC3668-type protein, an insect inhibitorysegment or fragment thereof, or any distinguishing portion thereof, arealso disclosed in this application. In certain embodiments, theprocessed product is selected from the group consisting of plant parts,plant biomass, oil, meal, sugar, animal feed, flour, flakes, bran, lint,hulls, processed seed, and seed. In certain embodiments, the processedproduct is non-regenerable. The plant product can comprise commodity orother products of commerce derived from a transgenic plant or transgenicplant part, where the commodity or other products can be tracked throughcommerce by detecting nucleotide segments or expressed RNA or proteinsthat encode or comprise distinguishing portions of a TIC3668-typeprotein.

Plants expressing the TIC3668 proteins can be crossed by breeding withtransgenic events expressing other toxin proteins and/or expressingother transgenic traits such as herbicide tolerance genes, genesconferring yield or stress tolerance traits, and the like, or suchtraits can be combined in a single vector so that the traits are alllinked.

TIC3668-type protein-encoding sequences and sequences having asubstantial percentage identity to TIC3668-type protein-encodingsequences can be identified using methods known to those of ordinaryskill in the art such as polymerase chain reaction (PCR), thermalamplification and hybridization. For example, the proteins of theTIC3668-type protein toxin class can be used to produce antibodies thatbind specifically to this class of proteins, and can be used to screenfor and to find other members of the class.

Further, nucleotide sequences encoding the TIC3668-type protein toxinclass (and reverse complement sequences) can be used as probes andprimers for screening to identify other members of the class usingthermal-cycle or isothermal amplification and hybridization methods.Specifically, oligonucleotides derived from sequences as set forth inany of SEQ ID NOs:52 through 61 can be used to determine the presence orabsence of a TIC3668-type transgene in a deoxyribonucleic acid samplederived from a commodity product. Given the sensitivity of certainnucleic acid detection methods that employ oligonucleotides, it isanticipated that oligonucleotides derived from sequences as set forth inany of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQID NO:61 can be used to detect a TIC3668, TIC3669, TIC3670, TIC4076,TIC4078, or TIC4260 transgene in commodity products derived from pooledsources where only a fraction of the commodity product is derived from atransgenic plant containing any of SEQ ID NO:52, SEQ LD NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, and SEQ ID NO:61. It is further recognized thatsuch oligonucleotides can be used to introduce nucleotide sequencevariation in each of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, and SEQ ID NO:61. Such “mutagenesis” oligonucleotides are usefulfor identification of TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, orTIC4260, amino acid sequence variants exhibiting a range of insectinhibitory activity or varied expression in transgenic plant host cells.

Nucleotide sequence homologs, e.g., insecticidal proteins encoded bynucleotide sequences that hybridize to each or any of the sequencesdisclosed in this application under stringent hybridization conditions,are also an embodiment of the present invention. The invention alsoprovides a method for detecting a first nucleotide sequence thathybridizes to a second nucleotide sequence, wherein the first nucleotidesequence (or its reverse complement sequence) encodes an insecticidalprotein or insecticidal fragment thereof and hybridizes under stringenthybridization conditions to the second nucleotide sequence. In suchcase, the second nucleotide sequence can be any of the nucleotidesequences disclosed in the TIC3668-type protein toxin class understringent hybridization conditions. Nucleotide coding sequenceshybridize to one another under appropriate hybridization conditions andthe proteins encoded by these nucleotide sequences cross react withantiserum raised against any one of the other proteins. Stringenthybridization conditions are known in the art and may vary according tothe desired application and outcome and may encompass a variety ofreagents and conditions. For instance, washes at higher temperaturesconstitute more stringent conditions. In certain embodiments,hybridization conditions of the present invention may comprise at leasthybridization at 42° C. followed by two washes for five minutes each atroom temperature with 2×SSC, 0.1% SDS, followed by two washes for thirtyminutes each at 65° C. in 0.5×SSC, 0.1% SDS; or hybridization at 68° C.,followed by washing at 68° C., in 2×SSC containing 0.1% SDS; orhybridization from 4 to 12 hours in 50% formamide, 1 M NaCl, and 1% SDSat 37C, and a wash in 0.1×SSC at 60C-65C.

One skilled in the art will recognize that, due to the redundancy of thegenetic code, many other sequences are capable of encoding such relatedproteins, and those sequences, to the extent that they function toexpress insecticidal proteins either in Bacillus strains or in plantcells, are embodiments of the present invention, recognizing of coursethat many such redundant coding sequences will not hybridize under theseconditions to the native Bacillus sequences encoding TIC3668. Thisapplication contemplates the use of these, and other identificationmethods known to those of ordinary skill in the art, to identifyTIC3668-type protein-encoding sequences and sequences having asubstantial percentage identity to TIC3668-type protein-encodingsequences.

This disclosure also contemplates the use of molecular methods known inthe art to engineer and clone commercially useful proteins comprisingchimeras of proteins from pesticidal proteins; e.g., the chimeras may beassembled from segments of the TIC3668-type proteins to deriveadditional useful embodiments including assembly of segments ofTIC3668-type proteins with segments of diverse proteins different fromTIC3668 and related proteins. The TIC3668-type protein class may besubjected to alignment to each other and to other Bacillus pesticidalproteins (whether or not these are closely or distantly relatedphylogenetically), and segments of each such protein may be identifiedthat are useful for substitution between the aligned proteins, resultingin the construction of chimeric proteins. Such chimeric proteins can besubjected to pest bioassay analysis and characterized for the presenceor absence of increased bioactivity and/or expanded target pest spectrumcompared to the parent proteins from which each such segment in thechimera was derived. The pesticidal activity of the polypeptides may befurther engineered for activity to a particular pest or to a broaderspectrum of pests by swapping domains or segments with other proteins orby using directed evolution methods known in the art.

Methods of controlling insects, in particular Lepidoptera or Coleopterainfestations of crop plants, with proteins from the TIC3668 toxinprotein class are also disclosed in this application. Such methods cancomprise growing a plant comprising an insect-, Coleoptera- orLepidoptera-inhibitory amount of a protein of the TIC3668 toxin proteinclass. In certain embodiments, such methods can further comprise any oneor more of: (i) applying any composition comprising or encoding aprotein of the TIC3668-type protein toxin class to a plant or a seedthat gives rise to a plant; and (ii) transforming a plant or a plantcell that gives rise to a plant with a polynucleotide encoding a proteinof the TIC3668-type protein toxin class. In general, it is contemplatedthat any protein in the TIC3668-type protein toxin class can be providedin a composition, provided in a microorganism, or provided in atransgenic plant to confer insect inhibitory activity againstLepidopteran or Coleopteran insects.

In certain embodiments, a recombinant polypeptide of the TIC3668-typeprotein toxin class is the insecticidally active ingredient of an insectinhibitory composition prepared by culturing recombinant Bacillus or anyother recombinant bacterial cell transformed to express a TIC3668-typeprotein toxin under conditions suitable to express and produce proteinsof the TIC3668-type protein toxin class. Such a composition can beprepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof such recombinant cells expressing/producing said recombinantpolypeptide. Such a process can result in a Bacillus or otherentomopathogenic bacterial cell extract, cell suspension, cellhomogenate, cell lysate, cell supernatant, cell filtrate, or cellpellet. By obtaining the recombinant polypeptides so produced, acomposition that includes the recombinant polypeptides can includebacterial cells, bacterial spores, and parasporal inclusion bodies andcan be formulated for various uses, including as agricultural insectinhibitory spray products or as insect inhibitory formulations in dietbioassays.

In one embodiment, to reduce the likelihood of resistance development,an insect inhibitory composition comprising one or more proteins fromthe TIC3668-type protein toxin class can further comprise at least oneadditional polypeptide that exhibits insect inhibitory activity againstthe same Lepidopteran or Coleopteran insect species, but which isdifferent from the TIC3668-type protein toxin. Possible additionalpolypeptides for such a composition include an insect inhibitory proteinand an insect inhibitory dsRNA molecule. One example for the use of suchribonucleotide sequences to control insect pests is described in Baum,et al. (U.S. Patent Publication 2006/0021087 A1). Such additionalpolypeptide for the control of Lepidopteran pests may be selected fromthe group consisting of an insect inhibitory protein, such as, but notlimited to, Cry1A (U.S. Pat. No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105,Cry1Ae, Cry1B (U.S. Patent Publication No. 10/525,318), Cry1C (U.S. Pat.No. 6,033,874), Cry1D, Cry1Da and variants thereof, Cry1E, Cry1F, andCry1A/F chimeras (U.S. Pat. Nos. 7,070,982; 6,962,705; and 6,713,063),Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry1-type chimeras such as,but not limited to, TIC836, TIC860, TIC867, TIC869 and TIC1100, Cry2A,Cry2Ab (U.S. Pat. No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9,Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC400, TIC800, TIC834,TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-184, AXMI-196, DIG-3, DIG-4, DIG-5,DIG-11, AfIP-1A and derivatives thereof (U.S. Patent Publication2014-0033361 A1), AfIP-1B and derivatives thereof (U.S. PatentPublication 2014-0033361 A1), PIP-1APIP-1B (U.S. Patent Publication2014-0007292 a1), PSEEN3174 (U.S. Patent Publication 2014-0007292 A1),AECFG-592740 (U.S. Patent Publication 2014-0007292 A1), Pput_1063 (U.S.Patent Publication 2014-0007292 A1), Pput_1064 (U.S. Patent Publication2014-0007292 A1), GS-135 and derivatives thereof (U.S. PatentPublication 2012-0233726 A1), GS153 and derivatives thereof (U.S. PatentPublication 2012-0192310 A1), GS154 and derivatives thereof (U.S. PatentPublication 2012-0192310 A1), GS155 and derivatives thereof (U.S. PatentPublication 2012-0192310 A1), SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Patent Publication 2012-0167259 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2012-0047606A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2011-0154536 A1, SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Patent Publication 2011-0112013 A1, SEQ ID NO:2 and 4and derivatives thereof as described in U.S. Patent Publication2010-0192256 A1, SEQ ID NO:2 and derivatives thereof as described inU.S. Patent Publication 2010-0077507 A1, SEQ ID NO:2 and derivativesthereof as described in U.S. Patent Publication 2010-0077508 A1, SEQ IDNO:2 and derivatives thereof as described in U.S. Patent Publication2009-0313721 A1, SEQ ID NO:2 or 4 and derivatives thereof as describedin U.S. Patent Publication 2010-0269221 A1, SEQ ID NO:2 and derivativesthereof as described in U.S. Pat. No. 7,772,465 (B2), CF161_0085 andderivatives thereof as described in W02014/008054 A2, Lepidopteran toxicproteins and their derivatives as described in US Patent PublicationsUS2008-0172762 A1, US2011-0055968 A1, and US2012-0117690 A1; SEQ ID NO:2and derivatives thereof as described in U.S. Pat. No. 7,510,878(B2), SEQID NO:2 and derivatives thereof as described in U.S. Pat. No.7,812,129(B1); and other Lepidopteran-inhibitory proteins known to thoseof ordinary skill in the art. Such additional polypeptide for thecontrol of Coleopteran pests may be selected from the group consistingof an insect inhibitory protein, such as, but not limited to, Cry3Bb(U.S. Pat. No. 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B,Cry34/35, 5307, Axmi184, Axmi205, AxmiR1, TIC407, TIC417, TIC431,TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10, eHIPs (U.S. PatentApplication Publication No. 2010/001714) and otherColeopteran-inhibitory proteins known to those of ordinary skill in theart.

In other embodiments, such composition/formulation can further compriseat least one additional polypeptide that exhibits insect inhibitoryactivity to an insect that is not inhibited by an otherwise insectinhibitory protein of the present invention to expand the spectrum ofinsect inhibition obtained. For example, for the control of Hemipteranpests, combinations of insect inhibitory proteins of the presentinvention can be used with Hemipteran-active proteins such as TIC1415(US Patent Application Publication No. 2013/0097735), TIC807 (U.S. Pat.No. 8,609,936), TIC834 (U.S. Patent Application Publication No.2013/0269060) and other Hemipteran-active proteins known to those ofordinary skill in the art. Additional polypeptides for the control ofColeopteran, Lepidopteran, and Hemipteran insect pests can be found onthe Bacillus thuringiensis toxin nomenclature website maintained by NeilCrickmore (on the world wide web at btnomenclature.info).

The possibility for insects to develop resistance to certaininsecticides has been documented in the art. One insect resistancemanagement strategy is to employ transgenic crops that express twodistinct insect inhibitory agents that operate through different modesof action. Therefore, any insects with resistance to either one of theinsect inhibitory agents can be controlled by the other insectinhibitory agent. Another insect resistance management strategy employsthe use of plants that are not protected to the targeted Coleopteran orLepidopteran pest species to provide a refuge for such unprotectedplants. One particular example is described in U.S. Pat. No. 6,551,962,which is incorporated by reference in its entirety.

Other embodiments such as topically applied pesticidal chemistries thatare designed for controlling pests that are also controlled by theproteins disclosed herein to be used with proteins in seed treatments,spray on, drip on, or wipe on formulations can be applied directly tothe soil (a soil drench), applied to growing plants expressing theproteins disclosed herein, or formulated to be applied to seedcontaining one or more transgenes encoding one or more of the proteinsdisclosed. Such formulations for use in seed treatments can be appliedwith various stickers and tackifiers known in the art. Such formulationscan contain pesticides that are synergistic in mode of action with theproteins disclosed, so that the formulation pesticides act through adifferent mode of action to control the same or similar pests that canbe controlled by the proteins disclosed, or that such pesticides act tocontrol pests within a broader host range or plant pest species that arenot effectively controlled by the TIC3668-type protein toxin class.

The aforementioned composition/formulation can further comprise anagriculturally-acceptable carrier, such as a bait, a powder, dust,pellet, granule, spray, emulsion, a colloidal suspension, an aqueoussolution, a Bacillus spore/crystal preparation, a seed treatment, arecombinant plant cell/plant tissue/seed/plant transformed to expressone or more of the proteins, or bacterium transformed to express one ormore of the proteins. Depending on the level of insect inhibitory orinsecticidal inhibition inherent in the recombinant polypeptide and thelevel of formulation to be applied to a plant or diet assay, thecomposition/formulation can include various by weight amounts of therecombinant polypeptide, e.g. from 0.0001% to 0.001% to 0.01% to 1% to99% by weight of the recombinant polypeptide.

EXAMPLES

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, specific structural and functionaldetails disclosed herein are not to be interpreted as limiting. Itshould be understood that the entire disclosure of each reference citedherein is incorporated within the disclosure of this application.

Example 1 Discovery of the TIC3668-Related Protein Toxin Class

Bacterial strains exhibiting distinctive attributes, e.g., inferredtoxicity, proteomic diversity, and morphological variations whencompared with each other, were identified and prepared for genomesequencing using methods well known in the art. A protein TIC3668 (SEQID NO:2) exhibiting inhibitory activity against Coleopteran insects inin vitro bioassays was discovered from a Brevibacillus laterosporus (B.laterosporus) strain EG5552. Other strains were also found to containproteins that resemble TIC3668. Polynucleotide segments encoding theseproteins were cloned, and inserted into a recombinant host strain totest for expression.

Thermal amplification primers were designed to amplify a full-lengthcopy of the gene from the total genomic DNA of different B. laterosporusbacterial strains, including EG5552. Separate thermal amplificationproducts (amplicons) were generated from each strain and these wereanalyzed for the presence of open reading frames that could encodeTIC3668-related proteins. Each amplicon was determined to have a singleopen reading frame, containing a translation initiation codon, followedin frame by a contiguous open reading frame, that terminated with anin-frame translation termination codon. The deducted amino acidsequences obtained from each of these additional different bacterialstrains are set forth respectively in SEQ ID NO:2 (TIC3668), SEQ ID NO:4(TIC3669), SEQ ID NO:6 (TIC3670), SEQ ID NO:8 (TIC4076), SEQ ID NO:10(TIC4078), SEQ ID NO:14 (TIC4346), SEQ ID NO:16 (TIC4826), SEQ ID NO:18(TIC4861), SEQ ID NO:20 (TIC4862), SEQ ID NO:22 (TIC4863). Theseamplicons were cloned into a recombinant Bacillus thuringiensis (Bt)plasmid expression vector downstream of a sporulation specificexpression promoter and transformed into an acrystalliferous Bt hostcell. The amplicons were also cloned into an E. coli expression system.The resulting recombinant strains were observed to express a recombinantprotein.

Example 2 Coleopteran Activity of TIC3668-Related Protein Toxin Class

This Example illustrates inhibitory activity exhibited by TIC3668-likeproteins against Coleoptera.

Protein preparations produced from recombinant bacteria as described inExample 1, for the full-length proteins of TIC3668, TIC3669, TIC3670,TIC4260, TIC4076 and TIC2462 were submitted for insect diet-overlaybioassays against Colorado Potato Beetle (Leptinotarsa decemlineata,CPB) and against at least one corn rootworm species. Known members ofcorn rootworm species are Diabrotica virgifera virgifera (Western CornRootworm, WCR), Diabrotica barberi (Northern Corn Rootworm, NCR),Diabrotica virgifera zeae (Mexican Corn Rootworm, MCR), Diabroticabalteata (Brazilian Corn Rootworm (BZR), Diabrotica undecimpunctatahowardii (Southern Corn Rootworm, SCR) and a Brazilian Corn Rootwormcomplex (BCR) consisting of Diabrotica viridula and Diabroticaspeciosa).

As demonstrated in Table 6, the results show that TIC3668, TIC3669,TIC3670, TIC4260, and TIC4076 exhibited mortality against corn rootworm.TIC2462 (SEQ ID NO:62 encoding SEQ ID NO:63), a protein closely relatedto the AXMI-209 protein (compared to TIC2462, >99% identical at theamino acid level, and exhibiting only two amino acid differences), didnot exhibit mortality against corn rootworm, thus distinguishing theactivity of the TIC3668-like protein toxin class from proteinsresembling AXMI-209. Surprisingly, mortality against Colorado PotatoBeetle, a species typically tested in bioassays as an indicator ofColeopteran activity, was not observed for any of the proteins tested.

TABLE 6 Observed Mortality against Coleopteran Insect Pests of ExemplaryProteins. Toxin Corn Rootworm CPB TIC2462 − − TIC3668, TIC3669,TIC3670 + − TIC4260, TIC4076 + − TIC4078 NT − TIC4346 + + TIC4826,TIC4861, TIC4862, NT NT TIC4863 + = Mortality observed − = Mortality notobserved NT = Not tested

Example 3 Mature Form of the TIC3668 Protein Toxin

This Example illustrates the presence of a membrane transiting peptideat the amino terminus of the native proteins within the TIC3668 proteintoxin class and the discovery of active mature toxin proteins of theTIC3668 protein toxin class.

Bioinformatic analysis using a SignalP program (Petersen, et. al (2011),Nature Methods, 8:785-786) of the amino acid sequence translation fromthe TIC3668 coding sequence (SEQ ID NO:1) predicted the presence of amembrane transiting segment corresponding to the N-terminal first 23amino acids.

Experiments were designed to confirm the presence of a membranetransiting segment within each member of the TIC3668-like protein toxinclass. TIC3668 was cloned into a Bt host cell behind a non-sporulationspecific Bt promoter. The resultant culture supernatants were tested forinsecticidal activity. Three forms of protein corresponding to TIC3668were recovered as a mixture from the supernatant. These differentfragments of less than full length TIC3668 protein were later determinedby mass spectrometry and N-terminal sequence analysis to contain attheir respective amino termini, either amino acid 16, 19, or 24, as setforth in SEQ ID NO:2. Only a small amount of these three truncated formsof TIC3668 were detected in the culture media. The most abundant form ofthe protein detected was observed to have at its amino terminus theserine at position 24, as set forth in SEQ ID NO:2. Concentrated andpurified protein from the culture supernatant exhibited bioactivityagainst WCR when tested in artificial diet bioassay.

Different expression constructs were created for identifying thesmallest peptide segment for each TIC3668-type protein exhibitinginsecticidal activity. These constructs were introduced into anacrystalliferous B. thuringiensis strain or an E. coli strain. Oneconstruct was designed for expression of the full length TIC3668protein, as set forth in SEQ ID NO:2 from amino acid 1 through 317, inan acrystalliferous strain of Bt. Constructs were designed forexpression of the full-length TIC3668 protein, and various shortervariant forms of the TIC3668 protein, in an E. coli expression systemhaving a carboxy terminal HIS tag sequence (HHHHAHHH). The constructsdesigned for expression in E. coli consisted of: (1) a constructdesigned to express the full length TIC3668 protein as set forth in SEQID NO:2 from amino acid position 1 through 317; (2) a construct designedto express a TIC3668 variant protein having from amino acid 16 through317 as set forth in SEQ ID NO:2; (3) a construct designed to express aTIC3668 variant protein from amino acid 24 through 317 as set forth inSEQ ID NO:2; (4) a construct designed to express a TIC3668 variantprotein from amino acid 26 through amino acid 317 as set forth SEQ IDNO:2; (5) a construct designed to express TIC3668 variant protein fromamino acid 28 through 317 as set forth in SEQ ID NO:2. Additionally aTIC3668 protein with an N-terminal 10-his tag and a TVMV (tobacco veinmottling virus) protease site (MHHHHHHHHHHGTETVRFQ) was obtained from anE. coli expression system to produce a TIC3668 protein with a start atresidue no. 24 as set forth in SEQ ID NO:2.

Protein was obtained from the supernatant of the Bt expression systemand subjected to mass spectrometry and N-terminal sequence analysis. TheBt expression system produced the predicted TIC3668 mature toxin fromacid 24-317 as set forth in SEQ ID NO:2. Protein was not observed in theE. coli supernatants. Protein was obtained from each of the respectiveE. coli expression constructs by osmotic shock to release proteins fromthe periplasm. Proteins produced from the constructs that were designedto contain amino acid 16 or 24 at the amino terminus of the less thanfull length protein were confirmed to contain these amino acids at theirrespective amino terminus. Protein produced from the construct designedto express the full length TIC3668 produced the mature length protein,containing the serine at position 24 as set forth in SEQ ID NO:2 at theamino terminus. Proteins produced from the constructs designed tocontain either amino acid 26 or amino acid 28 as set forth in SEQ IDNO:2 as the N-terminal amino acid each surprisingly contained only aminoacid 28 as the N-terminal amino acid, suggesting that processing thatmaintains amino acid number 24 as set forth in SEQ ID NO:2 at theN-terminus may be important for toxin stability.

Protein samples obtained from these expression system analyses weresubmitted for testing against Western Corn Rootworm larvae in insectdiet-overlay bioassays, as described in Example 2. Certain N-terminaltruncations from this study were determined to exhibit decreasedbioactivity. Specifically, it was observed that the insecticidalactivity was significantly reduced when the amino terminal amino acidwas 26 or 28, as set forth in SEQ ID NO:2. It can be extrapolated thatother TIC3668 protein family members that are N-terminally truncated tobe shorter than the mature protein (starting at amino acid residue no.24 for TIC3668, TIC:3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346,TIC4826, and TIC4863; starting at amino acid 13 for TIC4861; andstarting at amino acid 22 for TIC4862), are the shortest version of thetested TIC3668-type proteins to show insecticidal activity against WCR.All variants of TIC3668 of equal length or longer than the matureprotein showed high activity against WCR, even at relatively lowconcentrations. The data also demonstrates that the E. coli processingof TIC3668 varies by construct design.

Example 4 Synthesis of Genes Encoding TIC3668-Type Proteins forExpression in Plants

Nucleotide sequences encoding full-length and mature versions of aTIC3668 protein, a TIC3669 protein, a TIC3670, a TIC4076, TIC4078, aTIC4260 protein, a TIC4346 protein, a TIC4826 protein, a TIC4861protein, a TIC4862 protein, and a TIC4863 protein were designed.Nucleotide sequences encoding TIC3668, TIC3669, and TIC3670 weresynthesized according to methods generally described in U.S. Pat. No.5,500,365, avoiding certain inimical problem sequences such as ATTTA andA/T rich plant polyadenylation sequences while preserving the amino acidsequence of the native B. laterosperous protein. These nucleotidesequences are provided herein as SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:51 for the full-lengthsequences and SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, and SEQ ID NO:49for the mature sequences.

Example 5 Expression Cassettes for Expression of TIC3668-Type Proteinsin Plants

A variety of plant expression cassettes were designed with the sequencesas set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,and SEQ ID NO:51. Such expression cassettes are useful for transientexpression in plant protoplasts or transformation of plant cells.Typical expression cassettes were designed with respect to the eventualplacement of the protein within the cell. One set of expressioncassettes was designed in a manner to allow the protein to be translatedwith the native N-terminal segment. Another set of expression cassetteswas designed to allow the expression of the protein without theN-terminal segment (i.e., the mature length protein). Another set ofexpression cassettes was designed to have a transit peptide expressedin-frame and operably linked to the mature length toxin protein, toallow targeting to an organelle of the cell such as the chloroplast orplastid. All expression cassettes were designed to begin at the 5′ endwith a promoter which can be comprised of multiple contiguously linkedpromoter elements, enhancer elements or other expression elements knownto those of ordinary skill in the art to boost the expression of thetransgene. The promoter sequence was usually followed contiguously withone or more leader sequences 3′ to the promoter. An intron sequence wasprovided 3′ to the leader sequence to improve expression of thetransgene. A coding sequence for the toxin or transit peptide and codingsequence for the toxin was located 3′ of the promoter, leader and intronconfiguration. A 3′UTR sequence was provided 3′ of the coding sequenceto facilitate termination of transcription and provides sequencesimportant for the polyadenylation of the resulting transcript. All ofthe elements described above were arranged contiguously with oftenadditional sequence provided for the construction of the expressioncassette such as restriction endonuclease sites or ligation independentcloning sites.

Example 6 Transformation Vectors Containing TIC3668-Type ProteinExpression Cassette

Agrobacterium-mediated transformation vectors were constructed todeliver DNA to the plant genome that expresses the TIC3668, mTIC3668,TIC3669, mTIC3669, TIC3670, and mTIC3670 proteins. Expression cassetteswere cloned into suitable vectors between the Agrobacterium bordersequences such that they would be transferred to the genome of a hostplant cell by Agrobacterium hosts containing the construct vectors alongwith a selectable marker gene. More specifically, the restrictionfragment containing the entire cytosolic expression cassette encodingone of the proteins referenced above was cloned into an Agrobacteriumplant transformation vector. Similarly, the restriction fragmentcontaining the entire plastid targeted expression cassette was clonedinto an Agrobacterium plant transformation vector. The vectorscontaining the TIC3668-type protein expression cassettes (i.e.,untargeted cassette or targeted cassettes) are introduced intoAgrobacterium by electroporation or by tri-parental mating.

Expression cassettes containing artificial genes encoding TIC4076,TIC4078, TIC4260, TIC4346, TIC4826, TIC4861, TIC4862, and TIC4863, eachwith and without sequences encoding the N terminal 23 amino acidspresent in the native B. laterosperous open reading frame (amino acids1-23 as set forth in SEQ ID NO:2), are cloned into suitable vectorsbetween the Agrobacterium border sequences so that they are transferredto the genome of a host cell and tested for expression and bioactivityof the encoded protein.

Example 7 Coleopteran Activity of TIC3668-Type Proteins in Plants

This Example illustrates inhibitory activity exhibited by TIC3668-likeproteins against Coleoptera, such as corn rootworm larvae, whenexpressed in plants and provided as a diet to the respective insectpest.

R0 transgenic corn plants expressing TIC3668, mTIC3668, TIC3669,mTIC3669, TIC3670, and mTIC3670 proteins were produced using vectorscontaining the expression cassettes described in Example 5.

F1 transgenic corn plants were grown from seed produced by pollinatingears of non-transformed wild-type commercial germplasm plants withpollen from R0 transformants. After being transferred to soil in cagedpots, F1 plants were infested with neonate corn rootworm insects andgrown for 13 days under controlled conditions. Root damage ratings (RDR)were determined using the Oleson, et al. rating scale of 0-3, where 0means no injury and 3 means three or more nodes are pruned to within 1.5inches of the stalk (J. D. Oleson, Y-L. Park, T. M. Nowatzki, J. J.Tollefson, “Node-Injury Scale to Evaluate Root Injury by CornRootworms”, Journal of Economic Entomology, 98(1):1-8, 2005). Insectmortality was assessed by counting the number of third instar larvaeremaining at the end of the growth period.

In a first set of experiments, plants expressing the full-lengthTIC3668, TIC3669, and TIC3670 proteins were tested against WCR. Some ofthe events showed a statistical significant reduction in node injurycompared to the negative control with an average root damage rating(RDR) value between 2 and 2.5, but no commercially significant activitywas observed for the full-length proteins.

In a second set of experiments, mature proteins mTIC3668 (SEQ ID NO:23),mTIC3669 (SEQ ID NO:24), and mTIC3670 (SEQ ID NO:25), with or without achloroplast targeting peptide, were expressed in corn plants and testedagainst WCR. Significant WCR mortality was observed which each matureprotein. Each plant expressing mTIC3668, mTIC3669, and mTIC3670, in thepresence and absence of additional targeting sequences, showed astatistical significant reduction of node injury compared to thenegative control. FIG. 2 depicts the average RDR values for severalevents for mTIC3668 and mTIC3669 proteins and FIG. 3 depicts the averageRDR value for several events for mTIC3670 when expressed in F1 cornplants regardless of whether the protein was targeted to thechloroplast. “TS” in the event name of FIGS. 2 and 3 indicates thepresence of a targeting sequence. Commercially significant activity wasobserved for many of these events expressing mature proteins mTIC3668,mTIC3669, and mTIC3670.

Surprisingly, removal of the membrane transiting segment (amino acids1-23 as set forth in SEQ ID NO:2) from TIC3668-like proteins increasedthe efficacy against corn rootworm when expressed in corn plants. Whenexpressed in plants, the mature length TIC3668-like proteinsdemonstrated higher levels of insecticidal activity against Coleopteranpests than the full-length proteins.

Example 8 Insecticidal Activity of TIC3668-Related Proteins, Expressedin Corn, Against Cry3Bb1 Resistant WCR

This Example illustrates insecticidal activity exhibited by TIC3668-likeproteins against a strain of Western Corn Rootworm (WCR) that hasdeveloped resistance to the Bt toxin Cry3Bb1. F1 transgenic corn plantsexpressing mTIC3668, mTIC3669 or mTIC3670, produced using methods asdescribed in Example 7, were infested with 2000 WCR eggs of theHopkinton strain per plant.

The Hopkinton strain of Western Corn Rootworm (Diabrotica virgiferavirgifera LeConte) is a non-diapausing strain with field-evolvedresistance to Cry3Bb1 expressed in corn plants. The strain originatedfrom adult WCR samples obtained from fields that had been planted toCry3Bb1 corn for seven consecutive years. The population wasback-crossed with a non-diapausing WCR strain three times and selectedfor Cry3Bb1 resistance three times (Gassmann, et al. (2011) PLoS ONE6(7): e22629; Gassmann, et al. (2012) GM Crops Food 3(3): 235-244). Thecolony was obtained from the laboratory of Dr. Aaron Gassman at IowaState University, and is maintained by the Monsanto Biotech Entomologygroup in Chesterfield, Mo.

Following infestation, the WCR-Hopkinton strain eggs hatched within 48hours and the neonates began feeding on the roots. After 24 days, theroots were removed from the soil and corn root damage was evaluated asdescribed in Example 7, using the 0-3 scale. As shown in Table 7, theplants expressing mTIC3668, mTIC3669 and mTIC3670 were highly effectiveat protecting corn roots from damage in the presence of Hopkinton strainWCR neonates compared to control plants, thus overcoming the WCRresistance to the Cry3Bb1 toxin.

TABLE 7 Average RDR in Transgenic Corn Plants Infested with Cry3Bb1Resistant WCR Toxin N Average RDR (0-3) Standard Error mTIC3668 18 0.060.004 mTIC3669 15 0.05 1.82e−10 mTIC3670 14 0.05 1.95e−10 NegativeControl 6 2.14 0.24  N: number of plants evaluated

Example 9 Insecticidal Activity of TIC3668-Related Proteins, Expressedin Corn, Against Natural Infestation of WCR in Field Test Sites

This Example illustrates reduced root damage effectiveness exhibited bytransgenic corn plants expressing TIC3668-like proteins against naturalWCR infestations in Midwestern U.S. farm fields.

F1 transgenic corn plants expressing mTIC3668, mTIC3669 or mTIC3670,produced using methods as described in Example 7, were planted at fivelocations in Midwestern U.S. during late April to early May. Trials atthese locations relied on existing natural infestations for cornrootworm pressure. Root digging, for damage assessment, was completed bythe end of July. Rootworm damage was determined according to thenode-injury scale, as described in Example 7.

Results from the root dig trials indicated that under practicalconditions for farming in an open field, plants expressing mTIC3668,mTIC3669 and mTIC3670 were highly effective at protecting corn rootsfrom damage in the presence of natural corn rootworm pressure. Table 8shows the number of plants evaluated (N), the mean RDR and standarderror for test plants when locations are combined.

TABLE 8 Mean RDR in Transgenic Corn Plants Tested in Farm Field withNatural WCR Infestation Toxin N Mean RDR (0-3) Standard Error mTIC3668755 0.144 0.009 mTIC3669 1108 0.159 0.008 mTIC3670 1311 0.120 0.007Negative Control 362 1.426 0.047

Example 10 Lepidopteran Activity of TIC3668-Related Protein Toxin Class

This Example illustrates inhibitory activity exhibited by TIC3668-likeproteins against Lepidoptera, Protein preparations, as described inExample 1, for the full-length proteins of TIC3668, TIC3669 TIC3670,TIC4076, and TIC4078 were submitted for insect diet-overlay bioassaysagainst Black Cutworms (BCW, Agrotis ipsilon), Western Bean Cutworm(WBC, Striacosta albicosta), Corn Earworms (CEW, Helicoverpa zea),European Corn Borers (ECB, Ostrinia nubilalis), Sugarcane Borer (SCB,Diatraea saccharalis), Southwestern Corn Borer (SWC, Diatraeagrandiosella), cabbage looper (CLW, Trichoplusia ni), soybean looper(SBL, Chrysodeixis includes), and Fall Armyworm (FAW, Spodopterafrugiperda). Protocols and methods of preparing and performinginhibitory protein bioassays are known in the art.

Activity against certain Lepidopteran insect pests was observed forcertain TIC3668-type proteins as demonstrated in Table 9.

TABLE 9 Observed Stunting against Lepidopteran Insect Pests of ExemplaryProteins. Toxin ECB SWC BCW FAW CEW SBL TIC3668 ++ + NT − − −TIC3669 + + NT − − − TIC3670 ++ ++ NT − − + TIC4076 − +++ − − − +TIC4346 + + NT + + + TIC4078 NT NT NT − − + TIC4260, TIC4826 NT NT NT NTNT NT TIC4861, TIC4862, TIC4863 + = Stunting observed ++ = Stunting andmortality − = Mortality not observed NT = Not tested

Example 11 Lepidopteran Activity of TIC3668-Type Proteins in Plants

This example illustrates the inhibitory activity of the TIC3668-typeproteins to ECB, SWC, BCW, FAW, CEW, SBL when expressed in plants andprovided as a diet to respective insect pest.

Bioassays against Lepidopteran pests using plant leaf disks wereperformed similarly as described in U.S. Pat. No. 8,344,207 on TIC3668,TIC3669, and TIC3670 expressing R0 corn plants. The leaf damage rating(LDR) was assigned a rating score based upon the percent of the leafdisc devoured by the insect on a scale from 0 (0% eaten) to 11 (greaterthan 50%) eaten. Rating score steps increase incrementally by 5%. R0plants which do not contain insecticidal proteins served as negativecontrols. The cytosolic expression of the full-length TIC3668-typeprotein reduced feeding damage against CEW, FAW and SWC relative to theuntransformed control. Cytosolic expression of the TIC3670 proteinreduced feeding damage against SWC relative to the untransformedcontrol.

Example 12 Creation of the Collage Protein TIC4260

This Example teaches the creation of a novel gene sequence based on thefamily members of TIC3668. The amino acid variation from five of thenative TIC3668-type proteins was combined to create a novel collageprotein, TIC4260 (SEQ ID NO:12), that exhibits a different amino acidsequence diversity compared to the naturally occurring proteins. FIG. 1depicts the alignment of five native TIC3668-type proteins with TIC4260.Positions of sequence diversity are highlighted in gray shading in thissequence alignment. An artificial polynucleotide sequence wasconstructed (SEQ ID NO:11) that encodes the TIC4260 protein. The matureTIC4260 protein (mTIC4260, SEQ ID NO:28) is encoded by thepolynucleotide sequence as set forth in SEQ ID NO:43.

Similar alignments of other TIC3668-type proteins can be made in orderto create novel proteins exhibiting Lepidoptera and/or Coleoptera toxicactivity. These novel proteins are expressed, purified and testedagainst Lepidopteran and Coleopteran inspects in diet bioassays.Expression cassettes for these novel proteins are created andtransformed into plants to express these proteins to controlLepidopteran and Coleopteran pests of plants.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

It should be apparent to those skilled in the art that these different,improved sequence variations can be combined to create variants whichare also within the scope of this invention.

All publications and published patent documents cited in thespecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A recombinant polynucleotide molecule comprising a heterologouspromoter operably linked to a polynucleotide molecule encoding an insectinhibitory polypeptide, wherein said polypeptide comprises: (a) theamino acid sequence of SEQ ID NO:23; or (b) an amino acid sequencecomprising at least 80% identity to the amino acid sequence of SEQ IDNO:23; and wherein said recombinant polynucleotide molecule isfunctional for expression in a plant, a plant part, a plant tissue, aplant cell, a plant protoplast, a seed, or a bacterial cell.
 2. Therecombinant polynucleotide molecule of claim 1 comprising (a) thenucleotide sequence of SEQ ID NO:33 or SEQ ID NO:64 or (b) a nucleotidesequence comprising at least 80% identity to the nucleotide sequence ofSEQ ID NO:33, or SEQ ID NO:64.
 3. (canceled)
 4. An insect inhibitoryrecombinant polypeptide encoded by the recombinant polynucleotidemolecule of claim
 1. 5. The insect inhibitory recombinant polypeptide ofclaim 4, wherein said insect inhibitory recombinant polypeptidecomprises: (a) the amino acid sequence of SEQ ID NO:23; or (b) an aminoacid sequence comprising at least 80% identity to the amino acidsequence of SEQ ID NO:23.
 6. The insect inhibitory recombinantpolypeptide of claim 4, wherein said insect inhibitory recombinantpolypeptide exhibits inhibitory activity against an insect species ofthe order Coleoptera.
 7. The insect inhibitory recombinant polypeptideof claim 6, wherein said insect species of the order Coleoptera isWestern Corn Rootworm, Southern Corn Rootworm, Northern Corn Rootworm,Mexican Corn Rootworm, Brazilian Corn Rootworm, or Brazilian CornRootworm complex consisting of Diabrotica viridula and Diabroticaspeciosa.
 8. The insect inhibitory recombinant polypeptide according toclaim 4, wherein said insect inhibitory recombinant polypeptide exhibitsinhibitory activity against an insect species of the order Lepidoptera.9. The insect inhibitory recombinant polypeptide of claim 8, whereinsaid species of the order Lepidoptera is selected from the groupconsisting of European Corn Borer, Southwestern Corn Borer, BlackCutworm, Fall Army Worm, Corn Earworm, and Soybean Looper.
 10. A hostcell comprising the recombinant polynucleotide molecule of claim 1,wherein said host cell is selected from the group consisting of abacterial host cell and a plant host cell.
 11. An insect inhibitorycomposition comprising the recombinant polynucleotide molecule ofclaim
 1. 12. The insect inhibitory composition of claim 11, furthercomprising a nucleotide sequence encoding at least one other pesticidalagent that is different from said insect inhibitory polypeptide.
 13. Theinsect inhibitory composition of claim 12, wherein said at least oneother pesticidal agent is selected from the group consisting of aninsect inhibitory protein, an insect inhibitory dsRNA molecule, and anancillary protein.
 14. The insect inhibitory composition of claim 13,wherein said at least one other pesticidal agent exhibits activityagainst one or more pest species of the orders Lepidoptera, Coleoptera,or Hemiptera.
 15. The insect inhibitory composition of claim 14, whereinsaid at least one other pesticidal agent is selected from the groupconsisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1B, Cry1C, Cry1D,Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab,Cry3A, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35,Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400,TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901,TIC1201, TIC1415, VIP3A, and VIP3B protein.
 16. An insect inhibitorycomposition comprising the insect inhibitory recombinant polypeptide ofclaim 4 in an insect inhibitory effective amount.
 17. A method ofcontrolling a Coleopteran species pest, said method comprisingcontacting said pest with an insect inhibitory amount of the insectinhibitory recombinant polypeptide of claim
 4. 18. A seed comprising therecombinant polynucleotide molecule of claim
 1. 19. A commodity productcomprising the host cell of claim 10, said commodity product comprisinga detectable amount of said recombinant polynucleotide or an insectinhibitory recombinant polypeptide encoded by said recombinantpolynucleotide.
 20. A method of producing seed comprising therecombinant polynucleotide molecule of claim 1, said method comprising:(a) planting at least one seed comprising said recombinantpolynucleotide molecule; (b) growing plants from said seed; and (c)harvesting seed from said plants, wherein said harvested seed comprisessaid recombinant polynucleotide molecule.
 21. A recombinant vectorcomprising the recombinant polynucleotide molecule of claim
 3. 22. Therecombinant vector of claim 21, wherein said vector is selected from thegroup consisting of a plasmid, a bacmid, a phagemid, and a cosmid.
 23. Aplant resistant to insect infestation, wherein the cells of said plantcomprise the recombinant polynucleotide molecule of claim 1.